DEGRADATION OF NUCLEAR ONCOPROTEINS BY THE UBIQUITIN SYSTEM INVITRO
DEGRADATION OF NUCLEAR ONCOPROTEINS BY THE UBIQUITIN SYSTEM INVITRO
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DOI：
10.1073/pnas.88.1.139
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发表时间：
1991-01-01
影响因子：
11.1
通讯作者：
BRODEUR, GM
中科院分区：
文献类型：
--
作者：
CIECHANOVER, A;DIGIUSEPPE, JA;BRODEUR, GM
核癌蛋白是降解速度最快的细胞内蛋白之一。先前的研究表明泛素介导的蛋白水解系统参与了短寿命细胞内蛋白的周转。在本研究中，我们评估了泛素系统在降解由N-myc、c-myc、c-fos、p53和E1A基因编码的特定核癌蛋白中的潜在作用。这些核癌蛋白都是在体外合成的，在[S-35]蛋氨酸存在的情况下，通过转录适当的cDNA并翻译产生的mRNA。在无泛素细胞系统中监测标记蛋白的降解。ATP刺激所有蛋白质降解3到10倍。针对泛素激活酶E1的中和抗体完全抑制了降解，E1是泛素介导的蛋白水解级联反应中的第一个酶。此外，在每种情况下，通过添加亲和纯化的E1，可以恢复E1缺失裂解物的降解。这些数据表明泛素系统在体外介导了这些癌蛋白的降解。其他蛋白质的降解，如超氧化物歧化酶、细胞色素c、烯醇化酶、rna酶A和鸟氨酸脱羧酶，不是由泛素无细胞系统介导的。这表明这里研究的核癌蛋白具有特定的信号，通过这种蛋白水解途径靶向快速周转。此外，在无细胞系统中，各种E1A突变体在体内对降解的相对敏感性也保持不变，这表明泛素途径也可能在这些蛋白质的细胞降解中发挥作用。
Nuclear oncoproteins are among the most rapidly degraded intracellular proteins. Previous work has implicated the ubiquitin-mediated proteolytic system in the turnover of short-lived intracellular proteins. In the present study, we have evaluated the potential role of the ubiquitin system in the degradation of the specific nuclear oncoproteins encoded by the N-myc, c-myc, c-fos, p53, and E1A genes. Each of these nuclear oncoproteins was synthesized in vitro by transcription of the appropriate cDNA and translation of the resulting mRNA in the presence of [S-35]methionine. Degradation of labeled proteins was monitored in the ubiquitin cell-free system. ATP stimulated the degradation of all the proteins between 3- and 10-fold. The degradation was completely inhibited by neutralizing antibody directed against the ubiquitin-activating enzyme, E1, the first enzyme in the ubiquitin-mediated proteolytic cascade. Moreover, degradation in E1-depleted lysates could be restored in each case by the addition of affinity-purified E1. These data suggest that the ubiquitin system mediates the degradation of these oncoproteins in vitro. Degradation of other proteins, such as superoxide dismutase, cytochrome c, enolase, RNase A, and ornithine decarboxylase, is not mediated by the ubiquitin cell-free system. This suggests that the nuclear oncoproteins studied here possess specific signals that target then for rapid turnover by this proteolytic pathway. Furthermore, the relative sensitivity to degradation of various E1A mutants in vivo is also maintained in the cell-free system, suggesting that the ubiquitin pathway may play a role in the cellular degradation of these proteins as well.